This invention pertains to compositions and methods for mammalian cell culture in vitro, for example the culture of mammalian oocytes or embryos.
Embryo Cell Culture Media
Various cell culture media have been used to support the growth of mammalian cells in vitro. Many of these media are quite satisfactory in supporting the growth of certain cell types. Other cell types, however, have proven more difficult to support in vitro. There is a continuing need for improved media to support the growth of such cell types.
There is a particular need for improved media to support mammalian oocytes and embryos. A high percentage of embryos that are fertilized or transferred in vitro cease development-prematurely. The consequences are felt at both the economic and the human levels.
In the livestock industry, the value of in vitro-fertilized (IVF) embryos from genetically superior stock can be very high. Significant economic savings will result from methods that reduce the high rate of loss of bovine embryos.
Many forms of fertility treatments for humans involve the in vitro fertilization or transfer of oocytes or embryos. The success rates of human fertility treatments are not high. The low success rates impose substantial economic and emotional costs. Even incremental improvements in the success rate can be of substantial benefit. One of the many causes of the low overall success rate is the frequent failure of embryos to grow and develop properly in vitro. Improved media to better support embryo growth can not only enhance the success rate of fertility treatments, but ironically can also reduce the rate of multiple pregnancies resulting from the treatments. Because the overall success rate of current methods is low, practitioners often implant multiple embryos to increase the likelihood of pregnancy. Implanting multiple embryos increases the likelihood of multiple pregnancies as well. If each individual embryo were more likely to survive, then the perceived need to implant multiple embryos simultaneously would decline, and the rate of multiple pregnancies would decrease.
The development of bovine embryos has been supported by media containing inorganic salts, amino acids, carbohydrates, growth factors, and antioxidants. Development has been enhanced by co-culturing embryos with reproductive tissue cells and complex media containing sera and macromolecules. However, embryotoxic substances in these co-culture systems can interfere with the development of embryos. For example, it has been reported that ammonium and purines in the culture system have an embryotoxic effect on the pre-implantation development of bovine and murine embryos. Thus despite some improvements, the overall rate of successful development of embryos in vitro remains low. There is a continuing, unfilled need for improved media for the culture or co-culture of oocytes and embryos.
In vitro fertilization (IVF) and embryo transfer (ET) techniques for bovine oocytes and embryos have been used for both commercial and research purposes. Similar techniques have been used in infertility treatments for humans. Although several researchers have achieved high IVF success rates (xcx9c90%), only a small proportion (xcx9c20%) of in vitro fertilized zygotes develop to the morula and blastocyst stages. A large number of inseminated zygotes cultured in vitro cease development at the 8- to 16-cell stage, a time corresponding to embryonic genome activation. Relatively few morulae or blastocysts derived from IVF are suitable for ET.
Papers disclosing in vitro culture and co-culture systems for oocytes and embryos include the following: J. Lim et al., xe2x80x9cRoles of Growth Factors in the Development of Bovine Embryos Fertilized in vitro and Cultured Singly in a Defined Medium,xe2x80x9d Reprod. Fertil. Dev., 8:1199-1205 (1996); W. Eyestone et al., xe2x80x9cCo-culture of Early Cattle Embryos to the Blastocyst Stage with Oviductal Tissue or in Conditioned Medium,xe2x80x9d J. Reprod. Fert. 85:715-720 (1989); J. Thibodeaux et al., xe2x80x9cRole of Platelet-Derived Growth Factor in Development of in vitro Matured and in vitro Fertilized Bovine Embryos,xe2x80x9d J. Reprod. Fert. 98:61-66 (1993); J. Thibodeaux et al., xe2x80x9cStimulation of Development of In Vitro-Matured and In Vitro-Fertilized Bovine Embryos by Platelets,xe2x80x9d J. Anim. Sci. 71:1910-1916 (1993); J. Lim et al., xe2x80x9cA Serum-Free Medium for Use in a Cumulus Cell Co-Culture System for Bovine Embryos Derived from In Vitro Maturation and In Vitro Fertilization,xe2x80x9d Theriogenology 45:1081-1089 (1996); J. Lim et al., xe2x80x9cIntracytoplasmic Glutathione Concentration and the Role of xcex2-Mercaptoethanol in Preimplantation Development of Bovine Embryos,xe2x80x9d Theriogenology 46:429-439 (1996); J. Lim et al., xe2x80x9cA Continuous Flow, Perifusion Culture System for 8- to 16-Cell Bovine Embryos Derived from In Vitro Culture,xe2x80x9d Theriogenology 46:1441-1450 (1996); and J. Lim et al., xe2x80x9cPerifusion Culture System for Bovine Embryos: Improvement of Embryo Development by Use of Bovine Oviduct Epithelial Cells, and Antioxidant and Polyvinyl Alcohol,xe2x80x9d Reprod. Fertil. Dev., 9:411-418 (1997).
Nitric Oxide
The nitric oxide (NO) molecule controls a wide range of biological activities, including programmed cell-death (apoptosis), activation of guanylyl cyclase, interaction with superoxide anions to form peroxynitrite, regulation of glycolysis by modification of glyceraldehyde-3-phosphate dehydrogenase activity, control of the mitochondrial transport electron chain, the citric acid cycle, DNA synthesis, binding to the iron-sulphur center of enzymes, stimulation of ADP-ribosylation of proteins, production of arachidonic acid metabolites such as prostaglandin E2 and 5-hydroxyeicosatetraenoic acid, sperm motility and viability, and capacitation and hyperactivation of spermatozoa in vitro.
NO plays an important role in the regulation of various aspects of cell metabolism. During human pregnancy, NO is produced in the placenta, the decidua, and the endometrium. It has been reported that NO synthesis increases during pregnancy but decreases toward the end of gestation. A peak in NO synthesis has also been reported in the rat uterus during pregnancy. See Novaro et al., xe2x80x9cNitric oxide synthase regulation during embryonic implantation,xe2x80x9d Reprod. Fertil. Dev. 9:559-564 (1997). Early embryonic loss has been associated with local production of NO by decidual cells in the mouse uterus. See E. Haddad et al., xe2x80x9cEarly embryo loss is associated with local production of nitric oxide by decidual mononuclear cells,xe2x80x9d J Exp. Med., 182, 1143-1151 (1995).
Papers discussing nitric oxide and its role in reproductive biology include the following: L. McDonald et al., xe2x80x9cNitric Oxide and Cyclic GMP Signaling,xe2x80x9d PSEBM, 211:1-6 (1996); S. McCann et al., xe2x80x9cThe Role of Nitric Oxide in Reproduction,xe2x80x9d PSEBM, 211:7-15 (1996); Z. Katu{haeck over (s)}ixc4x87 et al., xe2x80x9cNitric Oxide Synthase: From Molecular Biology to Cerebrovascular Physiology,xe2x80x9d NIPS 9:64-67 (1994); A. Jablonka-Shariff et al., xe2x80x9cHormonal Regulation of Nitric Oxide Synthases and Their Cell-Specific Expression during Follicular Development in the Rat Ovary,xe2x80x9d Endocrinology 138:460-468 (1997); and M. Vega et al., xe2x80x9cExpression of Nitric Oxide Synthase (NOS) in Human Corpus Luteum (hCL) and the Role of Nitric Oxide (NO) on Luteal Steroidogenesisxe2x80x9d (abstract), Biol. Reprod., 54 (Suppl. 1):66 (1996).
A. Fukuda et al., xe2x80x9cProduction of nitric oxide from mouse embryo and effect of nitrite on mouse embryonic development in vitro,xe2x80x9d Biol Reprod 54:173 (abstr) (1996) reported that NO may have a regulatory role in preimplantation embryonic development in the mouse.
Hemoglobin (Hb) is widely known as the iron-containing molecule in red blood cells responsible for the transport of oxygen and carbon dioxide. It has recently been recognized that hemoglobin also binds nitric oxide with high affinity. Hemoglobin has been used in some experimental systems as a xe2x80x9csinkxe2x80x9d for nitric oxide that diffuses outside the cell during a process being studied.
The Invention
We have discovered that nitric oxide adversely affects the development of certain cells in vitro, such as the pre-implementation development of oocytes and embryos, particularly in a co-culture system. It has not previously been suggested that NO can be toxic in such systems. We have also discovered that the addition of a nitric oxide inhibitor such as hemoglobin to such systems eliminates this toxic effect, and promotes the maintenance, growth, and development of cells such as oocytes and embryos.
As is true of many other culture media, the standard embryo culture medium that we have used in our laboratory recently, modified bovine embryo culture medium (mBECM; J. Lim et al., Roles of growth factors in the development of bovine embryos fertilized in vitro and cultured singly in a defined medium. Reprod. Fertil. Dev. 8:1199-1205 (1996)), contains L-arginine (63.2 mg/L), which is a substrate for NO synthesis. (Arginine is normally included in the medium as it is has a variety of effects on cell metabolism, and is beneficial to the growth of embryos. Co-culture cells and serum can also be sources of arginine.) Thus embryos cultured in this medium may be able to synthesize NO for various biological events, as may cells such as cumulus-granulosa cells (CGs) used in embryo co-culture systems. We have found that, whatever its origin, NO in the culture medium can adversely affect embryonic development.
We have investigated the role of NO in the development of IVF bovine embryos up to the blastocyst stage. In a series of experiments, we examined the effects of adding a spontaneous NO releaser (sodium nitroprusside; SNP) and an NO scavenger (hemoglobin; Hb), to co-culture systems. In addition to these experiments, we also measured NO metabolites in both developing embryos and in developmentally arrested embryos. In other experiments, we attempted to determine whether CGs are responsible for the NO, and to determine the critical time in early embryonic development at which NO exerts its effects.
Following is a brief summary of the experimental methods and results reported in greater detail below.
In Example 1, embryos were cultured in a cumulus-granulosa cell (CG) co-culture system to which 0.008 or 0.04 mM of sodium nitroprusside (SNP), a spontaneous NO releaser, was added at 18 or 60 h post-insemination. Embryonic development was greatly (P less than 0.001) inhibited by the addition of SNP, regardless of the time of SNP addition and regardless of the SNP concentration. In Example 2,8-cell embryos were cultured singly in a defined medium, to which 0.0016, 0.008 or 0.04 mM of SNP was added. Development to the blastocyst stage greatly (P less than 0.001) decreased after addition of SNP, compared with controls. A higher (P less than 0.02) concentration of NO metabolites was found in developmentally-arrested embryos than in developing embryos at 144 h post-insemination (Example 3). In Example 4, embryos co-cultured with CGs reached the blastocyst stage at a significantly higher rate (P less than 0.02) after addition of hemoglobin (Hb, 1 xcexcg/ml). Pre-hatching development of embryos increased significantly (P less than 0.05) after addition of Hb at different time intervals (18, 60, or 144 h post-insemination) in Example 5. (xe2x80x9cHatchingxe2x80x9d refers to the point in development when the cytoplasm of the embryo increases to the point at which it breaks through its xe2x80x9couter membrane,xe2x80x9d the zona pellucida.) In the absence of CGs, embryonic development was not enhanced by adding Hb to the culture medium (Example 6). Pre-hatching development of 8-cell embryos derived from a Hb-containing culture system was not promoted by the further addition of Hb after transfer of the embryos to a defined and CG-free single-embryo culture system (Example 7). We concluded that NO, which may be secreted from CGs, inhibits the pre-hatching development of mammalian embryos fertilized in vitro, and that the use of an NO scavenger, Hb, in a co-culture system enhanced blastocyst formation.
We evaluated whether the effect of Hb on embryonic development was influenced by the time of the year (Example 8). In Example 9, we tested whether addition of an NO synthesis inhibitor, NG-Lxcfx89-nitro-L-arginine methyl ester hydrochloride (L-NAME), further augmented the positive effect of Hb. Bovine IVF-oocytes were used in our experimental models. In Examples 8 and 9, bovine IVF oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with either hemoglobin (Hb, 1 xcexcg/ml) or L-NAME (1 or 1000 nM) in a cumulus-granulosa cell co-culture system. In Example 8, a total of 1,675 cumulus-oocyte/zygote complexes were collected during a 9-month period and cultured to the blastocyst stage in mBECM, with or without Hb, after in vitro maturation and IVF. The effects of Hb addition and month of oocyte collection on embryonic development were significant (P less than 0.0024). Seasonal variation (P less than 0.0023) was detected in all developmental stages. However, addition of Hb to mBECM consistently enhanced embryonic development to the morula and blastocyst stages, regardless of the month. No significant differences were found in the interaction between Hb addition and month, except for the cleavage rate. Overall, a greater percentage of embryos developed to the 8-cell (P less than 0.0459), 16-cell (P less than 0.001), morula (P less than 0.0013), and blastocyst (P less than 0.0024) stages after addition of Hb than after no addition. In Example 9, addition of L-NAME to mBECM, supplemented with Hb, did not further stimulate pre-hatching development. The promoting effect of Hb on in vitro development of embryos was found to be highly repeatable over an extended period of time.